QD-0082-01 Pneumocystis Carinii Real Time PCR Kit
Contents
1. 10 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follow 171 181 0 4u1 1pl Oul Reaction Mix Enzyme Mix Internal Control 18 41 Master Mix Zul 18ul Extraction DNA Master Mix Reaction Plate Tube PCR Instrument X Only For Lightcycler 1 0 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls standards and sample prepared Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample n the number of reaction Reaction Volume Master Mix Volume Master Mix Volume Only for Lightcycler 1 0 Taxed aux TE a intemal control OO xa Mix completely then spin down briefly in a centrifuge 2 Pipet 18u Pneumocystis Carinii Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tubes Separately add 2ul DNA sample positive and negative controls to different reaction plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 37 C for 2 min 1 cycle 94 C 4 2 min 1 cycle 93 C for 5 sec bec for 30 sec 40 cycles Fluorescence is measured at 60 C 11 Data Analysis and Interpretation The following results are possible 1 A signal is detected i2. manufacturers You may use your own extraction systems or the commercial kit based on the yield For DNA extraction please comply with the manufacturer s instructions 10 2 Internal Control It is necessary to add internal control IC in the reaction mix Internal Control IC allows the user to determine and control the possibility of PCR inhibition Add the internal control IC 1ul rxn and the result will be shown in the VIC JOE channel 10 3 Quantitation The kit can be used for quantitative or qualitative real time PCR For performance of quantitative real time PCR standard dilutions must be prepared firstly as follows Molecular Grade Water is used as the dilution Dilution is not needed for performance of qualitative real time PCR detection 7 Take positive control 1x10 copies ml as the starting high standard in the first tube Respectively pipette 36ul Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards T i 5 4 110 copies ml 110 copies ml 1210 copies ml 14510 copies ml To generate a standard curve on the real time system all four dilution standards should be used and defined as standard with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer 7 B The positive control 1x10 copies ml contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination
3. A In addition the kit contains a system to identify possible PCR inhibition by measuring the VIC JOE fluorescence of the internal control IC An external positive control i 1x10 copies ml contained allows the determination of the gene load For further information please refer to section 10 3 Quantitation 5 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer 1 vial 1 8ml Pneumocystis Carinii Reaction Mix 1 vial 450ul PCR Enzyme Mix 1 vial 12ul Molecular Grade Water 1 vial 400ul Internal Control IC 1 vial 30ul Pneumocystis Carini Positive Control 1x10 copies ml 1 vial 30ul 6 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label 2 e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Reaction Mix should be stored in the dark 7 Additionally Required Materials and Devices e Biological cabinet e Real time PCR system e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Vortex mixer e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5ul 1000u1 e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks 8 Warnings and Pre
4. Revision No ZJ0003 C 4 Issue Date Aug e 2008 Pneumocystis Carinii Real Time PCR Kit Cat No QD 0082 01 For Use with LightCycler 1 0 LightCycler2 0 LightCycler480 Roche Real Time PCR Systems Pls ignore the Internal Control if you use the LightCycler 1 0 For In Vitro Diagnostic Use Only User Manual GENTAUR Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium 1 Intended Use Pneumocystis Carini real time PCR kit is used for the detection of Pneumocystis Carini in bronchial lavage sample or lung section sample by using real time PCR systems 2 Introduction Pneumocystis pneumonia PCP is a form of pneumonia caused by the yeast like fungus Pneumocystis jirovecii The causal agent was originally described as a protozoan and spelled P jiroveci and prior to then was classified as a form of Pneumocystis carinii a name still in common usage These names are discussed below As a result Pneumocystis pneumonia PCP has also been known as Pneumocystis jirovecii pneumonia and as Pneumocystis carinii pneumonia It is relatively rare in people with normal immune systems but common among people with weakened immune systems such as premature or severely malnourished children the elderly and especially AIDS patients in whom it is most commonly observed today PCP can also develop in patients who are taking immunosuppressant medications e g patients who have undergone solid organ transplantation and in patien
5. caution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink smoke in laboratory e Avoid aerosols 9 Sample Collection Storage and transport e Collect samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 10 Procedure 10 1 DNA Extraction DNA extraction buffer is supplied in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge befo
6. n channel FAM The result is positive The sample contains Pneumocystis Carinii DNA In this case the detection of a signal in channel VIC JOE Internal control is dispensable as high initial concentrations of Pneumocystis Carini1t DNA can lead to a reduced or absent fluorescence signal of the internal control competition 2 In channel FAM no signal is detected at the same time a VIC JOE signal from the internal Control appears The sample does not contain any Pneumocystis Carinii DNA It can be considered negative In the case of a negative Pneumocystis Carinii PCR the detected signal of the internal control rules out the possibility of PCR inhibition 3 Neither in channel FAM nor in channel VIC JOE is a signal detected A diagnostic statement can not be made Inhibition of the PCR reaction Gentaur Molecular Products Voortsiraat 49 1910 Kampenhout Belgium
7. re use 10 1 1 Bronchial lavage sample 1 Take 400ul sample in a tube and centrifuge the tube at 13000rpm for 2min Remove the supernatant and keep the sediment for processing 2 Add 50ul DNA extraction buffer in the tube sediment close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the DNA extracted and can be used for PCR template 10 1 2 Lung section sample 1 Wash the lung tissue with sterile saline for several times 2 Take 50mg sample in a tube add I ml sterile saline and grind the tissue into homogenate 3 Transfer the homogenate to a 1 5ml tube and centrifuge the tube at 13000rpm for 5min Remove the supernatant and keep the sediment for processing 4 Add 50ul DNA extraction buffer in the tube sediment close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 5 Incubate the tube for 10 minutes at 100 C 6 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template Attention A During the incubation make sure the tube is not open as the vapor will volatilize into the air and may cause contamination in case the sample is positive B The extraction sample should be used in 3 hours or stored at 20 C for one month C DNA extraction kits are available from various
8. ts who have undergone bone marrow transplantation 3 Principle of Real Time PCR The principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially Ct is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re open the reaction tube after the amplification 4 Product Description Pneumocystis Carini real time PCR kit contains a specific ready to use system for the detection of the Pneumocystis Carinii by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Pneumocystis Carinii DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified Pneumocystis Carinii DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQI1 DNA extraction buffer is available in the kit and bronchial lavage sample or lung section sample is used for the extraction of the DN
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